Impaired O2 unloading from stored blood results in diffusion-limited O2 release at tissues: evidence from human kidneys.

ABSTRACT: The volume of oxygen drawn from systemic capillaries down a partial pressure gradient is determined by the oxygen content of red blood cells (RBCs) and their oxygen-unloading kinetics, although the latter is assumed to be rapid and, therefore, not a meaningful factor. Under this paradigm, oxygen transfer to tissues is perfusion-limited. Consequently, clinical treatments to optimize oxygen delivery aim at improving blood flow and arterial oxygen content, rather than RBC oxygen handling. Although the oxygen-carrying capacity of blood is increased with transfusion, studies have shown that stored blood undergoes kinetic attrition of oxygen release, which may compromise overall oxygen delivery to tissues by causing transport to become diffusion-limited. We sought evidence for diffusion-limited oxygen release in viable human kidneys, normothermically perfused with stored blood. In a cohort of kidneys that went on to be transplanted, renal respiration correlated inversely with the time-constant of oxygen unloading from RBCs used for perfusion. Furthermore, the renal respiratory rate did not correlate with arterial O2 delivery unless this factored the rate of oxygen-release from RBCs, as expected from diffusion-limited transport. To test for a rescue effect, perfusion of kidneys deemed unsuitable for transplantation was alternated between stored and rejuvenated RBCs of the same donation. This experiment controlled oxygen-unloading, without intervening ischemia, holding all non-RBC parameters constant. Rejuvenated oxygen-unloading kinetics improved the kidney's oxygen diffusion capacity and increased cortical oxygen partial pressure by 60%. Thus, oxygen delivery to tissues can become diffusion-limited during perfusion with stored blood, which has implications in scenarios, such as ex vivo organ perfusion, major hemorrhage, and pediatric transfusion. This trial was registered at www.clinicaltrials.gov as #ISRCTN13292277.

Development of ex situ normothermic reperfusion as an innovative method to assess pancreases after preservation.

Static cold storage (SCS) is the standard method for pancreas preservation prior to transplantation; however, it does not permit organ assessment. Normothermic reperfusion (NR) is utilized clinically for other organs to assess viability. Our aim was to develop NR using normothermic machine perfusion technique to simulate reperfusion at the time of transplantation, enabling evaluation of oxygenated hypothermic machine perfusion (HMPO2) as a newer strategy to optimize pancreas preservation. 13 porcine pancreases procured after circulatory death were divided into 3 groups: 4 pancreases preserved using SCS, and 2 groups preserved by HMPO2 (n = 4 and n = 5, differing by type of preservation solution). Duration of perfusion or cold storage was 6 hours before the 1-hour assessment using NR. Outcome measures were perfusion characteristics, biochemistry and change in tissue water mass as oedema assessment. During NR, the HMPO2 groups demonstrated better perfusion characteristics, normal macroscopic appearances, decreased water mass and one HMPO2 group demonstrated a response to glucose stimulation. Conversely, the SCS group showed an increased water mass and developed early macroscopic appearances of oedema, interstitial haemorrhage and minimal portal outflow. This study suggests that ex situ assessment of pancreases by NR is promising, and that HMPO2 may be better than SCS.

CC-4066 therapy delivered to kidneys during cold storage and assessed with normothermic reperfusion is feasible and safe.

Introduction: Currently there is an urgent need to translate interventions that may be beneficial to marginal donor kidneys prior to transplant, to improve their quality from bench to bedside. This project investigated the effects of CC-4066, a potent dual inhibitor of cyclophilin proteins A and D, treatment during static cold storage (SCS) in a porcine model of renal ischemia-reperfusion injury (IRI) using Normothermic Reperfusion (NR). Materials and methods: Porcine kidneys and autologous blood were retrieved in pairs from a local abattoir (n = 7). One kidney from each pair was randomly allocated to treatment and one allocated to control and flushed with preservation solution containing CC-4066 or vehicle. After 7 h of SCS kidneys underwent 3 h Normothermic Reperfusion (NR) with autologous whole blood while perfusion characteristics and samples were collected. Results: Perfusion and metabolic parameters showed similar trends and no statistical differences were observed between the groups. IL-6 showed a significant increase over time but no significant difference between groups (p-value 0.009 and 0.14 respectively, two-way ANOVA). Oxygen consumption and lactate levels were similar between groups but there was increased vacuolation on histology in the control group. Conclusions: The addition of CC-4066 during SCS of kidneys is safe and feasible and has no adverse or detrimental effects on perfusion during assessment on NR. There was no difference in cytokine levels although there was a trend towards less vacuolation on histology in the treatment group.

Transplant and Recipient Factors in Prediction of Kidney Transplant Outcomes: A UK-Wide Paired Analysis.

BACKGROUND: In kidney transplantation, the relative contribution of various donor, procedure and recipient-related factors on clinical outcomes is unknown. Previous paired studies have largely focused on examining factors predicting early outcomes, where the effect of donor factors is thought to be most important. Here, we sought to examine the relationship between early and long-term outcomes in a UK-wide paired kidney analysis. METHODS: UK Transplant Registry data covering 24,090 kidney transplants performed between 2001-2018, where both kidneys from each donor were transplanted, were analysed. Case-control studies were constructed using matched pairs of kidneys from the same donor discordant for outcome, to delineate the impact of transplant and recipient factors on longer-term outcomes. RESULTS: Multivariable conditional logistic regression identified HLA mismatch as an important predictor of prolonged delayed graft function (DGF), in the context of a paired study controlling for the influence of donor factors, even when adjusting for early acute rejection. Prolonged DGF, but not human leucocyte antigen (HLA) mismatch, strongly predicted 12-month graft function, and impaired 12-month graft function was associated with an increased risk of graft failure. CONCLUSIONS: This study indicates prolonged DGF is associated with adverse long-term outcomes and suggests that alloimmunity may contribute to prolonged DGF by a mechanism distinct from typical early acute rejection.

Kidney Normothermic Machine Perfusion Can Be Used as a Preservation Technique and a Model of Reperfusion to Deliver Novel Therapies and Assess Inflammation and Immune Activation.

Ischaemia-reperfusion injury (IRI) is an inevitable process in transplantation and results in inflammation and immune system activation. Alpha-1 antitrypsin (AAT) has anti-inflammatory properties. Normothermic machine perfusion (NMP) can be used to deliver therapies and may help in assessing the effects of IRI and immunity. This study investigated the effects of AAT on IRI and inflammation in pig kidneys when administered during preservation, followed by normothermic reperfusion (NR) with autologous whole blood, as a surrogate for transplant. Two different models were used to deliver AAT or placebo to paired slaughterhouse pig kidneys: Model 1: 7-h static cold storage (SCS) + 3-h NR (n = 5 pairs), where either AAT (10 mg/ml) or placebo was delivered in the flush following retrieval; Model 2: 4-h SCS + 3-h NMP + 3-h NR (n = 5 pairs), where either AAT or placebo was delivered during NMP. Injury markers and cytokines levels were analysed in the perfusate, and heat shock protein 70 KDa (HSP-70) was analysed in biopsies. AAT delivered to kidneys showed no adverse effects on perfusion parameters. HSP-70 fold changes were significantly lower in the AAT group during NMP (P < 0.01, paired t-test) but not during NR. Interleukin-1 receptor antagonist (IL-1ra) fold changes were significantly higher in the AAT group during NR model 1 (p < 0.05, two-way ANOVA). In contrast to the AAT group, significant upregulation of interleukin-1 beta (IL-1ß) and interleukin-6 (IL-6) between t = 90 min and t = 180 min and interleukin-8 (IL-8) between baseline and t = 90 min was observed in the control group in NR model 2 (p < 0.05, Tukey's multiple comparison test). However, overall inflammatory cytokines and injury markers showed similar levels between groups. Delivery of AAT to pig kidneys was safe without any detrimental effects. NMP and NR provided excellent methods for comparison of inflammation and immune activation in the delivery of a novel therapy.

Abdominal multiorgan procurement from slaughterhouse pigs: a bespoke model in organ donation after circulatory death for ex vivo organ perfusion compliant with the 3 Rs (Reduction, Replacement & Refinement).

BACKGROUND: Advances in organ preservation, reconditioning and assessment have been driven by the increasing necessity to utilise organs from extended criteria donors, particularly donors after circulatory death. Research efforts in this area have aided translation of machine perfusion technology into clinical practice. Pigs are anatomically and physiologically similar to humans and are an excellent model. However, conducting large animal experimental research is challenging and typically limited by ethical and economic constraints. Here we describe a reproducible, cost-effective multi-organ abdominal procurement model of porcine organs from the slaughterhouse. METHODS: Domestic pigs are electrically stunned and exsanguinated following the standard abattoir process. Via a longitudinal midline incision, the thoracoabdominal viscera are removed en bloc by incising along the anterior vertebral plane. The abdominal organs are isolated, perfused and separated preserving their respective vasculature, allowing individual organ use for specific experiments. RESULTS: The warm ischaemic time is kept between 15-30 minutes. Using this highly protocolized procurement technique we have procured 12 livers, 162 kidneys and 12 pancreata for research, the majority of which have been utilized for ex situ perfusion experiments. CONCLUSIONS: We have described a reliable and reproducible procedure for abdominal multi-organ procurement from slaughterhouse pigs.

Evidence for Roux-en-Y Pancreatic Duct Drainage Versus Standard Anastomosis in Pancreatic Transplantation.

OBJECTIVES: Pancreatic transplantation is usually performed simultaneously with renal transplantation in the setting of end-stage nephropathy and type 1 diabetes. Surgical methods for dealing with exocrine secretions include bladder drainage, direct duodenojejunostomy and Roux-en-Y (ReY) enteric drainage. Roux-en-Y may confer an advantage over duodenojejunostomy because it distances enteric content from the transplant duodenal anastomosis. We examined the effect of enteric drainage method on transplant outcomes. METHODS: Data were obtained from the UK transplant registry on 2172 consecutive pancreatic transplants. Early graft loss was the primary endpoint. Secondary endpoints included return to theater, length of inpatient stay, readmission with pancreatitis, graft survival, and patient survival. RESULTS: There was no protective effect of ReY drainage (early graft loss, 4.6% vs 3.1%, P = 0.30; hazard ratio, 0.98; 95% confidence interval, 0.63-1.52; P = 0.91). There was a significant association between ReY and return to theater, reflecting either the technique or indication for ReY (multivariate odds ratio, 2.05; 95% confidence interval, 1.38-3.06; P < 0.01). The effect of transplant center on graft survival was assessed and adjusted for. CONCLUSIONS: There was no evidence of a protective benefit of ReY drainage over duodenojejunostomy, but there was an increased risk of return to theater.

Pancreatic Islet Changes in Human Whole Organ Pancreas Explants: What Can Be Learned From Explanted Samples?

BACKGROUND: Whole pancreas transplantation (Tx) is a successful treatment for type 1 diabetes resulting in independence from antidiabetic therapies. Transplant-related factors contributing to pancreatic islet failure are largely unknown; both recurring insulitis and pancreatitis have been implicated. The aim was to determine if cellular changes in islets and exocrine tissue are evident early in Tx, which could contribute to eventual graft failure using well-preserved tissue of grafts explanted from largely normoglycemic recipients. METHODS: Histological specimens of explants (n = 31), Tx duration 1 day-8 years (median 29 d), cold ischemia time 7.2-17.3 hours (median 11.1 h), donor age 13-54 years (median 38 y) were examined; sections were labeled for inflammation, islet amyloidosis, and tissue fibrosis, and morphometry performed on immunolabeled insulin and glucagon positive islet cells. Data were related to clinical details of donor, recipient, and features of Tx. RESULTS: Islet inflammation consistent with recurrent insulitis was not seen in any sample. Insulin-labeled islet cell proportion decreased with donor age (P < 0.05) and cold ischemia (P < 0.01) in explants from 26 normoglycemic patients; glucagon-labeled area proportion increased with cold ischemia (P < 0.05). Clinical pancreatitis was the explant reason in 12 of 28 normoglycemic cases. Exocrine fibrotic area/pancreas was variable (0.7%-55%) and unrelated to clinical/pathological features. Islet amyloid was present in 3 normoglycemic cases (donor ages 58, 42, and 31 y; Tx duration 8 y, 31 and 33 d, respectively). In 1 patient receiving antidiabetic therapy, the insulin-labeled area was reduced but with no evidence of islet inflammation. CONCLUSIONS: Explant histological changes after short-term Tx are similar to those seen in type 2 diabetes and occur in the absence of immunologic rejection without causing hyperglycemia. This suggests that factors associated with Tx affect islet stability; persistent deterioration of islet integrity and exocrine tissue fibrosis could impact on sustainability of islet function.

Isolated leptomeningeal carcinomatosis and possible fungal meningitis as late sequelae of oesophageal adenocarcinoma.

We describe a case of a 67-year-old man with known chronic obstructive pulmonary disease, type 2 diabetes mellitus, hypertension, osteoarthritis, previous history of excess alcohol intake, and oesophagectomy 3 years earlier for T3N0 adenocarcinoma, referred by his general practitioner with confusion, weight loss and several recent falls. CT of the chest, abdomen and pelvis revealed a right middle-lobe pulmonary embolism, while CT of the head revealed a communicating hydrocephalus. Lumbar puncture was performed, and empirical treatment for tuberculous and fungal meningitis was commenced. Unfortunately, he suffered a rapid neurological deterioration with markedly elevated cerebrospinal fluid (CSF) pressures, leading to an external ventricular drain. Cytological analysis of a CSF sample revealed a cellular infiltrate consistent with leptomeningeal carcinomatosis (adenocarcinoma), with the previous oesophageal malignancy the likely primary. He passed away 17 days after hospital admission. Prolonged culture of CSF later produced evidence of two distinct phaeomycotic moulds (Cladosporium sp and Exophiala sp), suggesting that fungal meningitis may also have contributed to the clinical picture.

Circadian profiling in two mouse models of lysosomal storage disorders; Niemann Pick type-C and Sandhoff disease.

Sleep and circadian rhythm disruption is frequently associated with neurodegenerative disease, yet it is unclear how the specific pathology in these disorders leads to abnormal rest/activity profiles. To investigate whether the pathological features of lysosomal storage disorders (LSDs) influence the core molecular clock or the circadian behavioural abnormalities reported in some patients, we examined mouse models of Niemann-Pick Type-C (Npc1 mutant, Npc1(nih)) and Sandhoff (Hexb knockout, Hexb(-/-)) disease using wheel-running activity measurement, neuropathology and clock gene expression analysis. Both mutants exhibited regular, entrained rest/activity patterns under light:dark (LD) conditions despite the onset of their respective neurodegenerative phenotypes. A slightly shortened free-running period and changes in Per1 gene expression were observed in Hexb(-/-) mice under constant dark conditions (DD); however, no overt neuropathology was detected in the suprachiasmatic nucleus (SCN). Conversely, despite extensive cholesterol accumulation in the SCN of Npc1(nih) mutants, no circadian disruption was observed under constant conditions. Our results indicate the accumulation of specific metabolites in LSDs may differentially contribute to circadian deregulation at the molecular and behavioural level.